Leptospirosis is a zoonosis of ubiquitous distribution caused by spirochetes. Leptospires exist either as saprophytic water-associated organisms or as animal pathogens that can survive in water. Previous works have demonstrated that both saprophytic and pathogenic leptospires are able to produce functional biofilms, which consist of a community of bacteria embedded in an extracellular matrix attached to a surface. This structure is believed to provide protection from environmental aggressiveness. In the present study, we analyzed the capacity of biofilm formation both of a a recent field isolate of Leptospira interrogans serovar Pomona obtained from an aborted swine fetus and of the saprophytic Leptospira biflexa serovar Patoc. We used light microscopy, immunofluorescence, and scanning electron microscopic examinations on glass and polystyrene plate models to evaluate the process in vitro. The ability to form bacterial aggregations in vivo was tested using pregnant guinea pigs infected with both strains. We obtained biofilms both on glass and plastic surfaces. Scanning electron microscopic analysis showed differences in the biofilm structure formed by both strains. L. interrogans serovar Pomona cell aggregations were observed in placental tissues by light microscopy. Biofilms and cell aggregations are consistent with the life of saprophytic strains in water and could help pathogenic strains to colonize the host and lead to abortion in pregnant animals.

Among the advances in microbiology that have taken place over the past 50 years, one of the most subtle has been the realization of the extent to which microbial growth and development occurs on surfaces in complex communities. Claude Zobell and colleagues noted that aquatic bacteria are more numerous on the solid surfaces of sample containers than as single suspended cells (19). Biofilm formation is an ancient and integral component of the prokaryotic life cycle, and is a key factor for survival in diverse environments. Recent advances have shown that biofilms arestructurally complex, dynamic systems with attributes of both primordial multicellular organisms and multifaceted ecosystems (5, 8). Biofilms are complex structures composed of millions of bacteria that work in a coordinated way either to avoid the phagocytic cells or resist the antibiotic action within the host (7).

Leptospirosis is a zoonosis of ubiquitous distribution caused by the spirochete of the genus Leptospira. Nowadays, leptospirosis is identified as a reemerging infectious disease that can be contracted either through direct contact with infected animals or from a contaminated environment due to the capacity of leptospires to survive in soil and water for long periods (16).

Singh et al. described a method based on a culture-independent direct amplification and sequencing of 165 subclones from community biofilm 16S rDNA that revealed that 20%of the identified bacteria corresponded to Leptospira spp. (16). Recently, Ristow et al. (15) described the formation of biofilms in saprophytic and pathogenic leptospires in vitro for the first time. The long-term colonization of the proximal renal tubules of rats by pathogenic leptospires is now believed to proceed via the formation of cell aggregates (2). Thus, biofilm formation may also play an important role in maintaining a chronic carriage of the pathogen Leptospira interrogans in animal reservoirs. For these reasons, we tested the ability of a recent local isolate of L. interrogans serovar Pomona obtained from an aborted pig to form biofilms in vitro and, as a preliminary study, its capacity to form bacterial aggregations in placental vessels of pregnant guinea-pigs.

Adherence to glass surfaces: Sterile microscope glass slides (25.4mm x 76.2mm x 1.2mm) and glass coverslips (18 x 18mm) were placed into sterile microscopy glass jars containing EMJH medium inoculated with L. interrogans serovar Pomona and L. biflexa serovar Patoc separately. The jar caps were sealed to avoid desiccation and cultures were grown at 30 °C. The experiment was performed in triplicate. The slides and the coverslips were removed from the jars at different time intervals (5, 15, 30 and 40 days), rinsed three times with phosphate buffered saline (PBS) 1X in order to eliminate the planktonic non-adherent bacteria and dried out. The slides were observed by light microscopy using the Whartin-Starry (W-S) staining method and Immunofluorescence (IF) using a polyvalent conjugate (USDA, USA) (1, 4). The slides were observed at a magnification of 1000X. The coverslips were used for Scanning Electron Microscopy (SEM) and fixed in 2.5 % glutaraldehyde at room temperature for 1 h. The fixed samples were gradually dehydrated in (V/V) 10 %, 30 %, 50 %, 70 %, 90 % and 100 % ethanol baths, desiccated and carbon evaporated. The samples were then observed under a JEOL JSM 6360LV field emission scanning electron microscope (JEOL Ltd, Japan).

Adherence to polystyrene surfaces: Sterile microscope coverslips (Thermanox, Nunc) were aseptically placed into 24-well polystyrene plates along with 200 µl of the EMJH media inoculated with the corresponding strain and incubated at different time points (5, 15, 30 and 40 days). Eighteen wells were used for the strains and the remaining six were used as control medium without inoculation. The cultures were stopped at the fixed times. The medium was removed and the coverslips were rinsed thoroughly as previously described with PBS 1X, W-S stained and observed by light microscopy and IF as previously described.

Animal experiments

In a previous work, the capacity of the swine isolate to induce abortion was studied in guinea pigs at different stages of pregnancy. Hartley pregnant guinea pigs that weighed 200 g were injected intraperitoneally with 1.5 x 107 of the swine isolate or the saprophytic strain in a final volume of 1 ml of EMJH. Animals were monitored daily for signs of illness. All the animals infected with the pathogenic strains aborted. Abortion was observed at the different stages of pregnancy studied (3). The presence of bacterial aggregations in placental vessels was tested only in 40-day pregnant guinea pigs. This stage of pregnancy was used according to the size of placental vessels. Three animals were inoculated intraperitoneally using 1.5 x 107 bacteria/ml of the swine isolate typified as L. interrogans serovar Pomona and other three were inoculated with L. biflexa serovar Patoc. The animals inoculated with the swine isolate were euthanized when the abortion occurred. Placenta tissue was extracted and analized for the presence of leptospiras by light microscopy. The placenta samples were fixed on 10 % formol and paraffinic tissue slides were prepared for the staining. The W-S staining technique was used and the samples were observed under a Leitz-Dialux microscope at a magnification of 1000X. All animal studies were approved by the Animal Research Care Committee of the National Institute of Agropecuarian Technology (INTA).

RESULTS

Biofilm formation on different abiotic surfaces

We compared the ability of a recent swine isolate of L. interrogans serovar Pomona and the saprophytic L. biflexa serovar Patoc to form biofilms on abiotic surfaces. We used light microscopy (Figure 1) and immunofluorescence (Figure 2) to observe the biofilm formation on glass surfaces and polystyrene coverslips at short (5 and 15 days) and long (30 and 40 days) incubation times. The swine isolate was able to aggregate and colonize most of the surface as compared with the saprophytic strain, in which case some remaining free bacteria could be observed at short incubation times (5 and 15 days) (Figures 1 and 2: A, B), whereas at long incubation times (30 and 40 days) both strains reached the same area of colonization (Figures 1 and 2: C, D). Both strains resisted the washes performed at the different incubation times. The results were the same when the strains were grown in polystyrene coverlips (data not shown).

Scanning electronmicroscopy was used to analyze the differences in the biofilm structures observed between both strains (Figure 3). The biofilm formed by L. interrogans swine isolate was able to cover most of the glass coverslip surface (Figure 3, 1A) as compared with that formed by L. biflexa that was set up dispersed on the glass surface (Figure 3, 2A and 2B). L. interrogans formed a dense structure where an intricate network of bacteria was observed (Figure 3, 1B and 1C). The structure formed by L. biflexa was less compact than that formed by L. interrogans and individual leptospires were observed on the surface (Figure 3, 2A).

The presence of cells aggregations was tested in 40-day pregnant guinea pigs. One of the three animals inoculated with the L. interrogans serovar Pomona aborted at 7 days pi. The necropsy was performed and placental samples were taken for W-S tissue staining. The other two animals aborted at 10 days pi and were euthanized following the same tissue treatment. None of the animals inoculated with L. biflexa serovar Patoc aborted, therefore, the animals were euthanized and placenta samples were taken.The W-S tissue staining revealed bacterial aggregations on the vascular endothelium of placental vessels only in the animals inoculated with the pathogenic isolate (Figure 4A). The animals inoculated with the saprophytic strain showed no bacterial aggregations but simple unique bacteria on the vascular endothelium (Figure 4B).

Leptospires can live in many different environmental conditions, such as water and soil, and the pathogenic species can also live in the microenvironment within the host. One of the mechanisms that could facilitate these adaptations is their capacity to form and maintain biofilms. Within a biofilm, bacteria become attached to a surface and form complex communities, which are able to interact with each other through intercellular communication and thus allow rapid adaptations to changing environments. The microorganisms within biofilms are notorious for their resistance towards the host immune response and antibacterial agents, as compared to their free-living planktonic counterparts(8). Twenty percent of bacterial biofilms are due toLeptospira spp., especially in water systems (16). Previous works have reported that Leptospira spp. can establish all the stages of a biofilm formation described by Hall-Stoodley et al. (8, 15).

According to these previous findings and because of the capacity of pathogenic leptospires to invade different mammalian tissues and to remain as a reservoir for further transmission, we decided to test the ability of a recent swine isolate to form biofilms in vitro and cell aggregations in vivo. The strain was isolated from a urine sample of an aborted pig and characterized serologically as L. interrogans serovar Pomona. In a previous work the strain pathogenicity was tested at different stages of pregnancy in a guineapig model and found that all the animals aborted, indicating that this strain was pathogenic and responsible for the pig abortion (3). In this work, the pathogenic isolate was compared with the saprophytic L. biflexa serovar Patoc. Light microscopy and immunoflurorescence showed that both strains formed layers of similar density in glass slides at long incubation times (40 days). At short incubation times (5 days), the pathogenic strain developed a thicker layer than that of the saprophytic strain (Figures 1 and 2, A and B). The plastic surfaces analyzed (polysterene slides and coverslips) rendered the same results (data not shown).The biofilms formed by both strains were resistant to the washes performed in both kinds of surfaces at both short and long incubation times. These results differ from those obtained by Ristow et al., who observed that L. interrogans was not resistant to the washes performed on the biofilm growing in a plastic surface at short incubation times. The authors suggested that this may be due to the fastidious growth rate of a recent pathogenic isolate growing in a static condition (15). Although a continuous-culture biofilm system was suggested to improve the bacterial attachment to the surface, the results shown in our study suggest that even when a static growth condition is used, a recent pathogenic low passage isolate may establish a complete biofilm structure if long incubation times are allowed and that this structure is resistant to washes. This is consistent with the SEM results obtained in our study where the biofilm structure observed in L. interrogans serovar Pomona covered almost all the surface and was thicker than the one formed by L. biflexa, where single leptospires were observed when long incubation time was tested.

Previous works have demonstrated that leptospires show evidence of social behavior, which may play a major role in leptospiral survival and transmission. Once the spirochetes reach natural collections of fresh water, they may detect the viscous milieu formed by cell capsules or biofilms from other organisms, upon which Leptospira cells aggregate (18). The animal experiments presented here allowed us to demonstrate that the recent pathogenic isolate L. interrogans serovar Pomona is able to aggregate in the placental vessel walls of pregnant guinea pigs, in contrast with the results obtained with the saprophyte strain. The presence of aggregates of pathogenic leptospires in placental vessels could be associated with the impairment of the mother-fetus interchange, which could be one of the reasons leading to abortion. The result is consistent with the fact that the different colonization factors that have been identified in L. interrogans serovar Copenhageni and serovar Lai genomes (11, 14) are absent in the L. biflexa genome (13). These factors correspond to different families of afimbrial adhesins that may contribute to the first steps of infection. The first family includes three paralogous genes, ligA, ligB and ligC, recently identified in L. interrogans and in Leptospira kirshneri, which codify for proteins with bacterial immunoglobulin-like (Big) repeat domains (10). The second family of leptospiral adhesin candidates consists of three integrin alpha-like proteins (LIC12259, LIC10021, and LIC13101) (11).These integrins are predicted to be integral membrane proteins, which would support their potential role in ligand-binding interactions on the leptospiral surface and may be involved in the establishment of bacterial aggregations observed in vivo.

Although more experiments are needed to prove this last hypothesis, in the present work we applied a successful model for the study of biofilm formation in vitro and cell aggregations in vivo. This model may be helpful in future studies aimed at understanding the mechanisms developed by pathogenic leptospires at the different stages of pregnancy that lead to abortion.